The invention relates to intact extended and super-extended DNA, methods of producing the extended DNAs and to efficient and rapid mapping of genes and other sequences employing various forms of extended DNA and DNA probes, particularly for visual mapping such as where fluorescent hybridization (comparable to in situ hybridization techniques) is employed. The remarkable sensitivity and resolution of direct visualization detection employing extended DNA permits rapid detailed studies of DNA rearrangements, such as translocations, and select determination of order and distance between DNA segments. Further, the invention concerns methods of controlling DNA extension to tailor to particular needs including long and short range mapping of DNA sequences.
A primary goal in developing a genome map is identification of genes and DNA sequences involved in disease states or disorders as well as in normal functions of the cell. The combined use of genetic and physical mapping of the human genome has proven useful in the placement of genes and/or molecular markers in reference to each other and in the cloning and identification of genes of biological and medical significance. This method of identification requires the use of genetic linkage data in a population to connect a disease or biological characteristic with molecular DNA markers. With this information, a physical map can be used to identify a gene of interest by its position in relation to the DNA markers.
The characterization of gene structure and genome organization has been greatly facilitated by the development of a number of physical mapping technologies. Among the first technologies developed were restriction mapping and DNA sequencing, which provide the highest resolution information, but are cumbersome when applied to large regions of DNA.
The development of pulsed-field gel electrophoresis and the use of rare cutting restriction enzymes has opened the door to restriction mapping in the megabase (Mb) size range. For the mapping of still larger regions of DNA, more recent developments of yeast artificial chromosomes (YACs) (Botstein et al., 1980) and radiation hybrid maps (Nakamura et al., 1987) have been useful. However, as the DNA regions to be mapped increase in size, fine structure resolution is generally sacrificed. Other techniques, such as the xe2x80x9cfingerprintingxe2x80x9d of repetitive elements (Litt et al. 1989), have been developed to overcome long range restriction mapping deficiencies.
A more global approach to mapping involves fluorescent in situ hybridization (FISH) to identify the position of probes on metaphase chromosomes (Weber et al. 1989). The use of this technique allows more rapid mapping of DNA probes with approximately 1 Mb resolution. For higher resolution mapping, the FISH technique has been applied to interphase nuclei (Evans et al. 1989). Since the DNA is less condensed in interphase nuclei than in metaphase chromosomes, resolution in the 50-100 kb range can be obtained. However, mapping the distance between two probes in three dimensional nuclei or compressed two dimensional nuclei is complex and requires large data sampling and probability calculations based on a random walk model (Coulson et al. 1988).
Long distance restriction maps of DNA regions have been generated using rare cutting restriction enzymes such as NotI (Poustka et al. 1987; Yagle et al. 1990; Barmeister et al. 1986). NotI linking clones, which encompass a NotI cleavable site, have been used to facilitate NotI mapping by identification of contiguous NotI fragments (Wallace et al. 1989). The use of frequent-cutting enzymes such as HindIII is not practical for mapping megabase-size DNA due to the complexity of the map.
Additional strategies for gathering physical linkage information on a still larger scale include the use of interspecific somatic cell hybrids, in which panels of rodent/human hybrid cell lines that retain various combinations of human chromosomes or parts thereof are used to localize probes to individual chromosomes or chromosomal regions (Ruddle et al. 1971). Radiation-induced hybrids, in which fragments of human chromosomes are retained in a rodent cell background (Goss et al. 1975) have also been employed.
Finally, fluorescent in situ hybridization (FISH) has become popular for determining approximate distances greater than 1 Mb between two or more probes on metaphase chromosomes (Lichter et al. 1990). FISH may also be used to a limited extent to determine the relative order of probes. For example, more closely linked probes along a 250 kb region have been hybridized to uncondensed DNA in interphase nuclei (Trask et al. 1989). While information concerning order may be obtainable with the method, there are some serious shortcomings; a major problem is that DNA in interphase nuclei is three dimensional. Labels that are not closely spaced or which are in reverse order to the observed order may be inaccurately determined because labels appear to be on top of one another, or because a twisted loop is viewed two-dimensionally.
A second shortcoming is the resolution. While improvements have been made, and the predicted resolution with FISH visualization is claimed to be about 10 kb, this range has not been substantiated, although resolution at 21 kbp has been reported (Heng et al. 1992). Higher resolution may be hampered because of the 3-D structure of the DNA, with lack of accessibility leading to poor resolution and difficulty in detection.
There have been reported attempts to reach levels of resolution around 10 kb; in one instance by expelling long 200 xcexcm loops from the nucleus (Wiegant et al. 1992), releasing chromatin fibers (Heng et al. 1992) or creating nuclear xe2x80x9chalosxe2x80x9d by extending DNA from which histones have been extracted (Lawrence et al. 1992). Although believed possible to extend resolution below 10 kb, there do not appear to be published data demonstrating that such resolution has been achieved.
There is, therefore, a need to develop physical mapping techniques that eliminate the tremendous amount of time and effort needed for restriction mapping and Southern blot hybridization and to increase the resolution limitation associated with FISH methods. New methods would allow microscopic visualization of cosmids or other DNA probes hybridized to an uncondensed fully extended DNA molecule, such as mapping of specific probes with high resolution exceeding 5 kb on YAC DNA. Direct visual mapping of repetitive DNA elements along a DNA strand would provide a significant improvement and alternative to restriction mapping and fingerprinting techniques.
The present invention addresses one or more of the foregoing or other problems encountered in chromosome mapping, particularly the complexity and inadequate resolution of current techniques. The present invention involves methods of preparing and visualizing DNA strands in linearized, that is, straight-line extended or xe2x80x9csuper-extendedxe2x80x9d forms as a means of mapping small or large DNA segments or even gene clusters.
The invention includes a simple, effective method to produce linear, one-dimensional DNA. The method provides extended and super-extended forms of DNA, the latter stretched to a length beyond the calculated xe2x80x9cunwoundxe2x80x9d length of native DNA. By comparison, the DNA from typical xe2x80x9cspreadsxe2x80x9d (Lehninger, 1970) is two-dimensional in the sense of exhibiting two directions in a single plane (including curves and winding). The form of DNA disclosed in the present invention is virtually straight (linear), has no contour and little, if any, randomness. It is thus visualized as extending in only one direction in a single plane. The invention therefore provides a procedure to stretch DNA into novel, linear, one-dimensional forms up to and beyond 0.34 xcexcm per kilobase pair. The DNA can be used as a target for hybridization of labelled probes to visually observe a high resolution map of probes along a single strand of DNA. Mapping resolutions as high as 1 kb are possible with the extended DNA. Super-extended DNA allows even further increases in resolution to at least 0.4 kb, although the extended form is practical for many applications.
Extended DNA is DNA stretched to a substantially one-dimensional form up to about 0.34 xcexcm per kilobase pair. Super-extended DNA is an essentially one-dimensional form stretched beyond what is commonly held to be the maximal length of fully extended DNA (0.34 xcexcm per kilobase pair). The inventors have shown super-extension of DNA up to at least 0.6 xcexcm per kilobase pair with no evidence of bond disruption. However, the inventors have observed that while the DNA length is stretched, the relative lengths remain generally proportional, i.e., the DNA stretches uniformly at least within limited areas. By varying the lengths of extended or super-extended DNA, rapid and accurate determination of order and distance between DNA segments is possible. Typically, such determinations employ variations of fluorescent in situ hybridization techniques (FISH), although other visualization techniques may be employed.
Stretching of DNA typically involves a gravitational streaming procedure such as allowing an aqueous sample of DNA to run down an appropriate surface; for example, a glass slide. Other procedures for stretching DNA in a linear fashion are possible, such as forced air streaming, the use of a charged field, or mechanical stretching, all of which are well known. In order to fix DNA to the surface over which it is stretched out, any of several fixing compositions may be employed; for example, formaldehyde, paraformaldehyde, glutaraldehyde, acids or alcohols. In a preferred embodiment, a DNA duplex is denatured, but the strands are not physically separated, thus allowing hybridization of probes, if desired, to the anchored strands. Lack of physical separation prevents division of the signal when labelled probes are employed, thus increasing sensitivity.
Accordingly, in one aspect of the practice of the invention, a novel DNA mapping technique employs extended or super-extended DNA hybridized with probes suitably labelled for detection. Standard FISH techniques, for example, permit direct visualization of the extended DNA under the microscope with resolutions as low as 0.4 kb. Other fluorescence visualization methods, or variations thereof, such as scintillography, radiography, chemoluminescence, fluorescence, etc. may also be utilized. Extended or super-extended DNA provides significantly increased resolution and greatly reduced preparation and analysis time.
There are several advantages of the disclosed DNA mapping method. For example, only one hybridization is necessary in order to determine the distance between two probes; thus, a restriction map of a DNA region (e.g., a YAC clone or genomic DNA) is not necessary for determining the distance between two probes. Additionally, very little DNA is required (e.g., 100 molecules (xcx9c1 ng) as opposed to xcexcg quantities). Information is acquired rapidly; for example, in a matter of days for some DNA mapping, rather than the weeks, months or even years required in some cases using standard mapping procedures. Chromosomal DNA can be mapped directly, thus avoiding mapping errors due to possible rearrangements in the clones. Mapping distances as large as 5 Mb between two probes is how feasible (considering size calculated from intact DNA that has been spread using other standard procedures). Using repetitive sequence probes, (e.g., L1, Alpha, or Alu repeats) a repeat map can be generated providing an alternative to the more involved, time consuming, and complex mapping of restriction sites and is a vast improvement over fingerprinting. Yet another advantage of the method is the ability to simultaneously determine the position of a specific sequence probe relative to a repeat map.
It should be mentioned that the length of fully extended, relaxed, duplex DNA is generally accepted as 0.34 xcexcm per kilobase pair (see, for example, Lehninger, 1970). This value is obtained from X-ray crystallographic data and is considered to be the maximum length of fully extended, double stranded relaxed xcex2-form DNA. Without deliberate stretching, fully relaxed DNA will not normally conform to a straight line. Clearly, however, the accuracy of measurements for straight-line DNA would be superior to measurements of DNA that winds around or overlaps itself. The present invention concerns a method of extending DNA as a straight line beyond its expected maximum of 3.4 xcexcm per kilobase pair and reaching at least 0.65 xcexcm per kilobase pair.
Extended or super-extended DNA can be hybridized with labelled probes to accurately determine distances between and order of the corresponding sequences. Direct visualization, such as the observation of multi-colored fluorescent probes under a microscope, provides virtually immediate mapping of DNA segments. Complete mapping is possible in a very short time with this technique; for example, only a few days are required for complete mapping of 15 cosmids covering a 500 kb region. This contrasts with estimates of 6-24 months to construct a similar genomic map by restriction mapping and fingerprinting. If there are gaps between some of the cosmids, additional cloning and mapping may be required by standard mapping. With the present invention, complete mapping is independent of gaps.
In more particular aspects, it has been discovered that stretching of DNA may be varied, much as a rubber band may be stretched to different degrees without breaking. By stretching a DNA thread to the maximum, which herein is understood to mean close to the point where covalent bonds are broken, short range distances along a gene or DNA sequence may be determined. Lesser degrees of stretching, e.g., linearization to a kilobase pair distance of about 0.34 xcexcm) are more suitable for longer range mapping.
As mentioned, the disclosed method of producing super-extended DNA involves stretching and then fixing DNA. One will generally employ cellular DNA, although DNA isolated from any source such as genomic DNA or cloned DNA may also be used.
Where cells are employed as the DNA source, the DNA is obtained by first disrupting the cell prior to stretching the DNA. Several methods of releasing the DNA from the cell may be employed including mechanical disruption, sonication, enzymatic degradation, hypotonic bursting, heat shock or cold shock. A convenient and preferred method is to treat the cellular DNA with a detergent. The detergent may be anionic, cationic or neutral and is preferably sodium dodecyl sulfate. The effect of the detergent is to dissolve the lipid material of the cell wall, denature and release proteins complexed to DNA thereby releasing the DNA.
The amount of sample DNA that is analyzed depends on the amount of data desired. For example, in order to map regions of DNA where hybridized DNA probes are observed under the microscope, very small amounts of DNA are required. Thus, a typical amount for cellular DNA samples comprises about 100 to 5,000 cells, i.e., 1-5 ng of cellular DNA.
In order to effectively stretch the DNA to an extended or super-extended form the DNA will be suspended in fluid. When cells are used as a source of a DNA sample this is generally no problem and intracellular disrupted suspensions may be used with or without further addition of fluid to the sample. Preferable fluids for suspension are basically aqueous, and can range from unbuffered to buffered solutions with phosphate, Tris or other salts. For highly concentrated samples of DNA or freeze dried samples, of course, a fluid should be added to create a suspension in order to affectively stretch out the DNA using the disclosed methods. While it is generally preferable to use aqueous solutions, other fluids such as alcohols and other organic solvents may be mixed with the aqueous media, preferably in relatively low amounts, e.g., 10% alcohol/water. Such mixtures would be expected to have the effect of altering the rate and amount of extension and may be tailored for particularly desired types of extension of the DNA molecule.
The disclosed extended or super-extended DNA is stretched by streaming the DNA over a supporting surface. Methods of DNA streaming include gravitational streaming, forced air or fluid streaming and stretching induced by charged electric fields, although other related methods such as mechanical stretching of DNA may also be employed. The gravitational streaming method involves tilting the supporting surface at an angle which will efficiently extend the DNA. Generally, the angle of tilt will be between about 20 to about 90 degrees from horizontal. The tilt angle employed will, of course, depend also on the surface employed for the support. Differences in surface material and surface tension of the DNA solution may affect the rate of streaming and the ultimate extension of the DNA molecule.
The support surface over which the DNA is gravitationally streamed should be relatively smooth and inert as to its reaction with the DNA. Glass or plastic are particularly preferred surfaces because these materials are used for microscope slides and several of the methods employing super-extended DNA utilize microscopic visualization. Other suitable support materials include ceramics, metal surfaces, cellophane or nitrocellulose and chemically coated surfaces.
Subsequent to stretching by gravitational streaming over a supporting surface the DNA is fixed to the surface. Fixing is generally by a chemical treatment although other means of attachment to the surface may be employed; for example, by UV crosslinking. Cellular DNA, after release from the cell, is surrounded by proteins. The fixative will therefore act to anchor the DNA to the proteins and the proteins will be anchored to the surface. Direct anchoring of DNA to the surface is also possible. In the case of purified DNA without protein, the addition of protein may facilitate the DNA anchoring process. Numerous commonly available fixatives may be employed, such as methanol/acetic acid mixtures, formaldehydes, glutaraldehyde or heat. Alcohol and common mineral or organic acids are effective and inexpensive fixatives. Methanol and acetic acid mixtures are particularly preferred for fixing cellular DNA to a solid surface.
In preferred practice, extended or super-extended DNA is in duplex form and is fixed to the matrix over which it has been stretched. The fixing conditions employed are such that the DNA duplexes are denatured but do not appear to separate. Denaturation allows hybridization of probes, yet the two strands are anchored sufficiently to keep them from appearing as two strands. The resulting signal, typically fluorescence labels, is at least twice as bright as probe hybridized to a single isolated strand.
In one aspect of the invention, a method of DNA mapping is disclosed. The mapping method generally includes the steps of obtaining a DNA sample, forming extended or super-extended DNA, hybridizing the DNA with a suitable probe and then determining the position of the probe on extended DNA. By employing extended or super-extended DNA and labelling with a probe that is easily visualized, relative probe position and order are readily and quickly determined. The high resolution obtained is possible because the super-extended DNA is substantially linear with distance greater than 0.34 xcexcm per kilobase pair. The distance per kilobase pair may be extended as far as 0.65 xcexcm. Since the extension results in a substantially linear form, that is, a flat single-dimension rather than a 3-dimensional matrix the straight line form of the DNA allows easy measurement of appropriate labels that show position and order. DNA probes may be labelled with direct or indirect forms of fluorescent labels, radiolabels, luminescent or calorimetric labels. Labels may include conjugates to nucleotides such as biotin, DNP, digoxin or the like.
Two aspects of the sensitivity of the disclosed method are quite surprising. These appear to be related to the disclosed procedure for stretching and fixing DNA. Sensitivity of detection of a DNA size in the range of 5 kb is readily obtained and sensitivity down to 0.4 kb has been determined. The high sensitivity is greater by at least an order of magnitude than standard FISH can attain and appears due to the DNA being extended rather than compressed in the nucleus. For hybridized target DNA that is compacted in a small space, there is little room for binding of proteins such as the avidin used in the detection system. While the bound probe in a compressed nucleus may have a greater capacity to bind the detection proteins, steric hindrance appears to effectively hinder binding. On the other hand, the hybridized extended DNA has protein binding sites far from each other with little contact; thus, there is less or negligible steric hindrance and a greater capacity to bind the labelled proteins.
The second aspect of sensitivity obtained with the present invention relates to the small number of molecules required to generate mapping information. Theoretically, a single cell may be lysed and its DNA stretched and analyzed. This is in stark contrast with the more than one million cells required for a single Southern blot analysis.
Visualization directly under the microscope to determine relative order and distance between DNA segments will be referred to herein as DIRVISH DNA mapping, which is understood to mean direct visual hybridization DNA mapping. Measurement of physical distances that represent map distances has been referred to previously.
Labelled probes may be hybridized to cellular DNA using methods well known to those of skill in the art. In preferred embodiments, detection of labelled probes is by direct visualization; for example, by direct visualization of fluorescent probes. Various probes may be differentially labelled, for example, with one, two, three, or more different fluorescent probes that are visualized under the microscope as different colors. Visualization need not be by fluorescent microscopy, however, and other methods, such as light microscopy or scintillography could be used for detection.
It is contemplated that DNA desired for mapping will most usually be cellular DNA. Cellular DNA will be duplexed DNA and is preferably treated with a chromatin-disassociating agent prior to extension. In some cases it may be beneficial to denature the DNA duplexes prior to extension.
In genome mapping, DIRVISH DNA mapping can be used to rapidly map the position and order of either restriction fragments, cosmids or YACs. With the development of 12 color fluorescent probe labelling, it is possible to map 12 probes, such as restriction fragments, simultaneously.
A common strategy to identify a gene responsible for a genetic disease is to use genome mapping to narrow the region of interest to xcx9c1 Mb. The 1 Mb region is then analyzed by a variety of restriction mappings and Southern blot hybridizations using several different probes to identify potential disease causing deletions, inversions or insertions that may allow one to pinpoint the location of the disease gene. DIRVISH DNA mapping can map such a region in a normal cell within one week, and will rapidly detect and pinpoint alterations in the gene from a small number of patient""s cells as part of a routine screen. An example of this is the NF-1 gene of neurofibromatosis patients which can be identified by three different deletions of 190 kb, 40 kb and 11 kb, all of which are easily mapped by DIRVISH DNA mapping.
A rapid and simple screen of rearrangements in known disease associated genes is possible using DIRVISH DNA mapping using a very small cell sampling. Detection of rearrangements can be diagnostic and prognostic; for example, the detection of deletions in the NF-1 gene in the diagnosis of patients with neurofibromatosis type 1, or the detection of abl-bcr rearrangements in patients as a means of diagnosis and prognosis based on pinpointing the breaksite junction. The detection of rearrangements in the Duchenne""s muscular dystropy gene would serve as a diagnosis of that disorder.
In further aspects, the present invention concerns a kit for mapping DNA. The kit includes an apparatus for extending or preparing super-extended DNA and also contains any of numerous probes suitable for hybridization with segments of a DNA of interest. The probes may be labelled with a variety of labels, for example, fluorescein, biotin or the like.
An apparatus such as that shown in FIG. 1 for preparing extended or super-extended DNA will include a tiltable surface preferably adjustable to an angle between 0 to 90 degrees from horizontal. The angle may be adjusted mechanically by hand; for example, by a step-adjustable lock mechanism or alternatively adjustable through electronic means such as chip powered batteries or externally supplied power. The apparatus may include mechanisms for automatically varying the tilt angle over a specified period of time. The apparatus may also provide for handling of several samples simultaneously with the same or different tilting angles.
Yet another aspect of the invention includes novel extended forms of DNA produced by gravitationally stretching DNA (see FIG. 2A). Such extended DNAs are unique forms of DNA with several differences in properties, compared to the three-dimensional, relatively compact forms of DNA normally encountered in the cell (see FIG. 2B). The extended or stretched forms of DNA have an interkilobase pair distance equal to or less than 0.34 xcexcm. Super-extended DNA has an interkilobase pair distance of greater than 0.34 xcexcm per kilobase pair and up to or beyond 0.65 xcexcm.
The invention therefore provides a much improved mapping technique with many practical and clinical applications. Clinically, the method better pinpoints translocation breakpoints using small sample size, in some cases even a single cell. Structural changes in genes may be detected in small or heterogenous samples. Examples include the analysis of amnionic fluid or p53 gene deletions in tumor samples where cosmid probes cover a 400 kb region, alternating colors with one end labelled with another probe, or mapping regions of replication which can provide a physical reference point and map for detecting position of origins.
Additional applications include quick mapping of restriction fragments in the genome or cosmid, mapping orientation of cosmid to any other probe site, mapping one cosmid to another cosmid (overlap or gap), mapping restriction fragments in YACs, YAC to YAC (overlap or gap), or orientation of YAC to another probe site, mapping sites of exons  greater than 0.4 kb in a gene using cDNA probes, mapping sites of PCR products such as inter-alu PCR, producing unique patterns for specific regions, mapping unique patterns for specific regions, mapping repeats such as Alu or Line or Alpha in specific regions, producing unique patterns as a map for ubiquitous repeats (Alu or Line) mapping on YACs directly from yeast cells or genome fragments from somatic or radiation hybrids, mapping unique probes from NotI linking or jumping library or related systems, detecting rearrangements; for example, inversions, deletions, etc. in cosmids, YAC clones by comparison to genomic DNA or mapping rearrangements in genes for diagnostic significance.